Shortly after the first strains of dengue virus (DEN) were recovered fifty years ago, serial passage of DEN serotype 1 or 2 in mouse brain rapidly selected for neurovirulent mutants that concomitantly exhibited significant attenuation for humans. This rapid coordinate aquisition of mouse neurovirulence and attenuation for humans suggested that the genetic basis of these two phenotypes might be related. We had previously employed full-length DEN4 cDNA to construct a viable intertypic dengue type 1 or type 2 chimera that contained the C-PreM-E or only the PreM-E genes of DEN1 or DEN2 substituting for the corresponding genes of DEN4. Studies of the mouse neurovirulent mutant of the DEN2 NGC strain identified Glu406 to Lys substitution in the envelope glycoprotein (E) as responsible for the acquisition of dengue type 2 virus mouse neurovirulence. This observation suggested that it might be possible to create an attenuating mutation in dengue viruses of all four serotypes as part of a general strategy for vaccine development. In an attempt to evaluate this strategy, we constructed DEN3/DEN4 chimeras that contained DEN3 C-PreM-E genes and expressed DEN3 antigenic specificity. A full-length DNA template of DEN3/DEN4 was prepared by in vitro ligation and used for transcription. Progeny virus recovered from RNA transfected mosquito C6/36 cells exhibited DEN3 antigenic specificity as determined by reaction with monoclonal antibodies. Gel electrophoresis of virus-infected cell lysates yielded the predicted viral protein pattern; i.e., DEN3 C, PreM and E and DEN4 nonstructural proteins. Two amino acid substitutions, Thr435Leu and Glu406Lys analogous to mutations that, respectively, confer mouse neurovirulence on DEN4 or DEN2, were introduced into DEN3 E. Mutant chimera containing Thr435Leu substitution, which ablates the potential glycosylation site sequence, produced E identical in size to wild type DEN3 E, indicating that the glycosylation site is normally not used. The Thr435Leu mutant was not neurovirulent. In contrast, intracerebral inoculation of sucking mice revealed that the mutant chimera containing the Glu406Lys substitution was neurovirulent, whereas its chimeric parent or parental DEN3 was not.